We thank D. L. Court for providing plasmid pSim6 for the recombineering experiments. This work was supported by HKU CRCG Seed Funding Programme for Basic Research and by a grant (HKU 7485/06M) from the Research Grant Council of Hong Kong to JDH. AD acknowledges support from the PROBACTYS programme, grant CT-2006-029104 in an effort to define genes essential for the construction of a synthetic cell.